Complete Mitochondrial Genome of the Western Capercaillie Tetrao Urogallus (Phasianidae, Tetraoninae)
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Zootaxa 4550 (4): 585–593 ISSN 1175-5326 (print edition) https://www.mapress.com/j/zt/ Article ZOOTAXA Copyright © 2019 Magnolia Press ISSN 1175-5334 (online edition) https://doi.org/10.11646/zootaxa.4550.4.9 http://zoobank.org/urn:lsid:zoobank.org:pub:12E18262-0DCA-403A-B047-82CFE5E20373 Complete mitochondrial genome of the Western Capercaillie Tetrao urogallus (Phasianidae, Tetraoninae) GAËL ALEIX-MATA1,5, FRANCISCO J. RUIZ-RUANO2, JESÚS M. PÉREZ1, MATHIEU SARASA3 & ANTONIO SÁNCHEZ4 1Department of Animal and Plant Biology and Ecology, Jaén University, Campus Las Lagunillas, E-23071, Jaén, Spain. E-mail: [email protected] 2Departamento de Genética, Facultad de Ciencias, Universidad de Granada, Avda. Fuentenueva, 18071 Granada, Spain. 3BEOPS, 1 Esplanade Compans Caffarelli, 31000 Toulouse, France 4Department of Experimental Biology, Jaén University, Campus Las Lagunillas, E-23071, Jaén, Spain 5Corresponding author Gaël Aleix-Mata: [email protected] ORCID: 0000-0002-7429-4051 Francisco J. Ruíz-Ruano: [email protected] ORCID: 0000-0002-5391-301X Jesús M. Pérez: [email protected] ORCID: 0000-0001-9159-0365 Mathieu Sarasa: [email protected] ORDCID: 0000-0001-9067-7522 Antonio Sánchez: [email protected] ORCID: 0000-0002-6715-8158 Abstract The Western Capercaillie (Tetrao urogallus) is a galliform bird of boreal climax forests from Scandinavia to eastern Sibe- ria, with a fragmented population in southwestern Europe. We extracted the DNA of T. urogallus aquitanicus and obtained the complete mitochondrial genome (mitogenome) sequence by combining Illumina and Sanger sequencing sequence da- ta. The mitochondrial genome of T. urogallus is 16,683 bp long and is very similar to that of Lyrurus tetrix (16,677 bp). The T. urogallus mitogenome contains the normal 13 protein-coding genes (PCGs), 22 transfer RNAs, 2 ribosomal RNAs, and the control region. The number, order, and orientation of the mitochondrial genes are the same as in L. tetrix and in other species of the same and other bird families. The three domains of the control region contained conserved sequences (ETAS; CSBs), boxes (F, E, D, C, B, BS box), the putative origin of replication of the H-strand (OH) and bidirectional promoters of translation (LSP/HSP). Key words: control region, mitogenome, Phasianidae, Tetrao urogallus Introduction The Order Galliformes contains about 290 species, approximately 10% of which are listed as globally Endangered or Critically Endangered (del Hoyo et al. 1994; Hosner et al. 2016; IUCN 2017). Traditionally, the species of this order are divided into seven families: Megapodiidae, Cracidae, Odontophoridae, Numididae, Phasianidae, Meleagrididae, and Tetraonidae (del Hoyo et al. 1994). The family Phasianidae, with more than 150 species, is distributed throughout the world (Johnsgard 1986; Fuller & Garson 2000; Fuller et al. 2000). The genus Tetrao (subfamily Tetraoninae) includes two extant species, T. urogallus Linnaeus and T. urogalloides Middendorff. The Western Capercaillie (Tetrao urogallus) is the best-known species of this genus and is found typically in boreal climax forests from Scandinavia to eastern Siberia, but also in a fragmented population in southwestern Europe (Storch 2007). In this latter region, it is present in the Pyrenees (France, Andorra and Spain) and in the Cantabrian Mountains of Spain (de Juana 1994). Using morphological characteristics, up to 12 T. urogallus subspecies have been described (de Juana 1994), although some are not well supported by mitochondrial DNA analyses (Liukkonen-Anttila et al. 2004). The two capercaillie subspecies of southwestern Europe, T. urogallus aquitanicus Ingram of the Pyrenees and T. urogallus cantabricus Castroviejo of the Cantabrian Mountains, are both considered Threatened in small-scale assessments (Canut et al. 2004; Obeso 2004; Rodríguez- Muñoz et al. 2007; Charra & Sarasa 2018). Accepted by P. Rasmussen: 12 Nov. 2018; published: 29 Jan. 2019 585 Although scientific information on demography over the last 20 years is scarce (Gée et al. 2018), the biology and ecology of T. urogallus is quite well-known, and information is available on reproduction, population and/or subspecific variations, and genetics (Rodríguez-Muñoz et al. 2007; Mollet et al. 2015; Fameli et al. 2017; Kowalczyk et al. 2017; Rutkowski et al. 2017). Genetic studies have analyzed several DNA markers, most of them mitochondrial DNA sequences [12s rRNA, 16s rRNA, cytochrome oxidase I (CoI), cytochrome B (CytB), control region (D-loop)] and several microsatellites (Segelbacher et al. 2008; Lucchini et al. 2001; Dimcheff et al. 2002; Kerr et al. 2009; Pérez et al. 2011). At present, a complete mitogenome sequence is not available for either of the two Tetrao species. However, the mitogenome of the Black Grouse, Lyrurus tetrix Linnaeus, a closely related species previously included in genus Tetrao, has been described (Li et al. 2016). In this study, we sequenced and described the complete mitochondrial genome of T. urogallus aquitanicus, which we then compared with that of L. tetrix. Materials and methods DNA extraction, sequencing, and mitogenome assemblage. Genomic DNA was extracted from liver tissue of an individual of T. urogallus aquitanicus using the DNeasy Blood & Tissue Kit (Qiagen). For genome sequencing, 3 µg of genomic DNA were used for the construction of a library with 750 bp fragments. This library was used in Illumina® Hiseq™ 2000 paired-end sequencing with 100 bp reads. Two Gbp of sequences were obtained (coverage about 1.5X). We assembled the sequence for the T. urogallus mitogenome using the MITObim v1.8 program (Hahn et al. 2013) with the "--quick" option and with the default mismatch of 15%. For this purpose, we randomly selected one million read pairs with seqTK (https://github.com/lh3/seqtk) and used as a reference the mitogenome of Lyrurus tetrix (accession number KF955638.1; Li et al. 2016). A region containing the D-loop and the gene Nd6 of the mitochondrial genome was not recovered completely and so was amplified by PCR using the same T. urogallus sample and sequenced. For PCR amplification the primer pair Pro+ (5’- ACCATCAGCACCCAAAGCTG-3’) and Phe- (5’-AAGCATTTTCAGTGCTTTGCTT-3’) were used (Haring et al. 2000). Sequences were analyzed with Bioedit (version 7.0.9.0) (http://www.mbio.ncsu.edu/BioEdit/ bioedit.html). The annotation of the T. urogallus mitogenome was fulfilled using web-based services MITOS (http://mitos.bioinf.uni-leipzig.de/help.py) (Bernt et al. 2013) and tRNA scan-SE (http://lowelab.ucsc.edu/ tRNAscan-SE/) (Lowe & Eddy 1997). The annotations of protein coding genes (PCGs), transfer RNAs (tRNAs) and rRNA genes were refined by comparing manually with the L. tetrix mitogenome (Li et al. 2016). The circular drawing of the mitogenome was carried out using the OrganellarGenomeDRAW tools (http://ogdraw.mpimp- golm.mpg.de/) (Lohse et al. 2013). Results and discussion Genome organization and gene arrangement. The mitochondrial genome of T. urogallus was assembled and submitted to GenBank (accession number MG583885). It is composed of 13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and the non-coding region (D-loop) (Table 1, Fig. 1). The mitogenome of T. urogallus is 16,683 bp long, similar to the L. tetrix mitogenome of 16,677 bp (Li et al. 2016); both have a percentage of identity of 94.70%. The total base composition of the T. urogallus mitochondrial genome is 30.15% A, 30.55% C, 13.62% G, and 25.67% T; the A-T content is 55.82%, which is also very similar to the L. tetrix base composition and A-T content (30.37% A, 30.42% C, 13.38% G, 25.83% T; A-T content 56.20%) (Li et al. 2016). Protein coding genes (PCGs). The total length of the 13 PCGs of T. urogallus was 11,392 bp (68.23% of the total length of the mitogenome); the similarities with the 13 PCGs of L. tetrix is very high (varying between 96.10% of the Nd1 and 92.66% of the Nd6) (Table 2). The main differences affect the Nd6 gene of L. tetrix, which has an 18-bp nucleotide deletion and gives rise to a smaller protein; this deletion is absent from the same gene in T. urogallus and other Phasianidae species (Nishibori et al. 2001; Guan et al. 2010; Kan et al. 2010; Shen et al. 2010; Li et al. 2016). In addition, as in other bird species including L. tetrix, the Nd3 gene of T. urogallus has an extra nucleotide in position 174 (mitogenome nt 10,855, C) that is not translated (Mindell et al. 1998; Li et al. 2016). 586 · Zootaxa 4550 (4) © 2019 Magnolia Press ALEIX-MATA ET AL. TABLE 1. Gene organization of the Tetrao urogallus mitogenome. Gene Strand Nucleotide positions Size (bp) Anticodon Intergenic nucleotide D-loop H 1–1141 1141 tRNAPhe H 1142–1208 67 TTC 12S rRNA H 1209–2171 963 tRNAVal H 2172–2244 73 GTA 16S rRNA H 2245–3858 1614 tRNALeu (uur) H 3859–3932 74 TTA Nd1 H 3944–4918 975 11 tRNAIle H 4919–4992 74 ATC tRNAGln L 4999–5069 71 CAA 6 tRNAMet H 5069–5137 69 ATG -1 Nd2 H 5138–6176 1039 tRNATrp H 6177–6253 77 TGA tRNAAla L 6260–6328 69 GCA 6 tRNAAsn L 6333–6405 73 AAC 4 tRNACys L 6408–6473 66 TGC 2 tRNATyr L 6473–6543 71 TAC -1 COI H 6545–8095 1551 1 tRNASer (ucn) L 8087–8161 75 TCA -9 tRNAAsp H 8164–8232 69 GAC 2 COII H 8234–8917 684 1 tRNALys H 8919–8989 71 AAA 1 Atp8 H 8991–9155 165 1 Atp6 H 9146–9829 684 -10 COIII H 9829–10,612 784 -1 tRNAGly H 10,614–10,681 68 GGA 1 Nd3 H 10,682–11,033 352 tRNAArg H 11,035–11,103 69 CGA 1 Nd4L H 11,104–11,400 297 Nd4 H 11,394–12,771 1378 -7 tRNAHis H 12,772–12,840 69 CAC tRNASer H 12,842–12,906 65 AGC 1 tRNALeu H 12,908–12,978 71 CTA 1 Nd5 H 12,979–14,796 1818 CytB H 14,801–15,943 1143 4 tRNAThr H 15,946–16,014 69 ACA 2 tRNAPro L 16,017–16,086 70 CCA 2 Nd6 L 16,093- 16,614 522 6 tRNAGlu L 16,616–16,683 68 GAA 1 Intergenic nucleotide: denotes the number of overlapping nucleotides (negative values) or the number of spacer nucleotides (positive values) between two consecutive genes.